Journal of Helminthology (2002) 76, 37±43 DOI: 10.1079/JOH200198

Failure to infect laboratory rodent hosts with human isolates of Rodentolepis (= ) nana

M.G. Macnish1, U.M. Morgan1, J.M. Behnke2 and R.C.A. Thompson1* 1WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections and Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia: 2School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

Abstract Confusion exists over the species status and host-specificity of the tapeworm Rodentolepis (= Hymenolepis) nana. It has been described as one species, R. nana, found in both humans and rodents. Others have identified a subspecies; R. nana var. fraterna, describing it as morphologically identical to the human form but only found in rodents. The species present in Australian communities has never been identified with certainty. Fifty one human isolates of Rodentolepis (= Hymenolepis) nana were orally inoculated into Swiss Q, BALB/c, A/J, CBA/ CAH and nude (hypothymic) BALB/c mice, Fischer 344 and Wistar rats and specific pathogen free (SPF) hamsters. Twenty four human isolates of R. nana were cross-tested in flour beetles, Tribolium confusum. No adult worms were obtained from mice, rats or hamsters, even when immunosuppressed with cortisone acetate. Only one of the 24 samples developed to the cysticercoid stage in T. confusum; however, when inoculated into laboratory mice the cysticercoids failed to develop into adult worms. The large sample size used in this study, and the range of techniques employed for extraction and preparation of eggs provide a comprehensive test of the hypothesis that the human strain of R. nana is essentially non-infective to rodents.

Introduction There is confusion in the literature regarding both the nomenclature and host specificity of R. nana. A taxo- Rodentolepis (= Hymenolepis) nana is the most common nomic revision of hymenolepidids with armed rostellae cestode infecting humans worldwide (Smyth, 1994). (hooks present) was proposed by Spasskii in 1954 Estimates of the number of human infections range (Vaucher, 1992) in which Hymenolepis nana was reclassi- from 20 million (Andreassen, 1998) to 50±75 million fied under the genus Rodentolepis and this classification is (Pawlowski, 1984; Crompton, 1999) worldwide. Humans currently recognized by some cestode taxonomists may also be infected with the rat tapeworm, Hymenolepis (Czaplinski & Vaucher, 1994). The genus , but this is considered rare and usually Weinland 1858 now only contains hymenolepidid species accidental (Schantz, 1996; Andreassen, 1998) and almost with unarmed rostellae (hooks absent) and the type always occurs in children (Tena et al., 1998). species is the rat tapeworm Hymenolepis diminuta (Czaplinski & Vaucher, 1994). Despite the revised *Author for correspondence nomenclature being adopted by some taxonomists Fax: (61 8) 9310 4144 (Spasskii, 1994; Czaplinski & Vaucher, 1994) Rodentolepis E-mail: [email protected] nana is almost universally referred to as Hymenolepis nana 38 M.G. Macnish et al.

Table 1. Inoculation of SPF mice and flour beetles with human isolates of Rodentolepis (=Hymenolepis) nana.

Samples yielding Number of Number of cysticercoids (beetles) Samples yielding SPF mice or beetles Species mice or beetles Sex Age samples tested or adult worms (mice) adult worms

Swiss Q Mouse 85 Male 4±5 wks 51 0 0 BALB/c Mouse 98 Male 4±5 wks 51 0 0 BALB/c nude² Mouse 5 Male 4±5 wks 1 0 0 Flour beetles T. confusum 110 ND ND 11a 1* 0 Flour beetles T. confusum 130 ND ND 13b 00 a Eggs obtained by NaCl flotation. b Direct faecal feeding. * One sample yielded 73 cysticercoids dissected from five Tribolium confusum beetles infected via ad libitum feeding of eggs on filter paper. ² Hypothymic model. ND, not determined.

in the medical literature. (cf. Arme & Pappas, 1983; Materials and methods Pawlowski, 1984; Smyth & McManus, 1989; Smyth, 1994; Baily, 1996; Schantz, 1996; Lloyd, 1998; Andreassen, Source of parasites 1998). A laboratory isolate of R. nana was kindly provided by In Australia, human infections with Rodentolepis (= Dr Akira Ito, Gifu University, Japan in two forms: (i) eggs Hymenolepis) nana are most common in communities in in PBS; and (ii) approximately 30 flour beetles, Tribolium tropical regions. The prevalence of R. nana has risen from confusum, infected with the cysticercoid stage of R. nana. 20% (Meloni et al., 1993) to approximately 55% in these The isolate has been passaged through laboratory mice same communities (Reynoldson et al., 1997) within the for more than 20 years. Human faecal samples were last decade. There is uncertainty about the transmission collected from the north-west region of Western Australia of R. nana in these endemic areas where human infection over a period of three years. Positive identification of is common. It is not well understood whether the `strain/ infection was carried out using the ZnSO4 flotation species' of R. nana present in the north-west of Western technique (Voge, 1970). Eggs were usually extracted from Australia is found in rodent and human hosts or whether faecal samples using the NaCl flotation method (Voge, humans harbour their own form of this parasite. For 1970) prior to inoculation into specific pathogen free example, R. nana has been described as being infective to (SPF) mice, rats and hamsters (tables 1 and 2). Other egg both humans and rodents (Yamaguti, 1959). Others have extraction techniques used in this study are outlined (see concluded that the rodent form is a subspecies, R. nana Infection methods tested using human isolates). var. fraterna, morphologically indistinguishable from the human form and only infective to rodents (cf. Baer & Tenora, 1970; Pawlowski, 1984; Schantz, 1996; Lloyd, Infection of mice and beetles using a laboratory reference strain 1998). In the present study, some of these uncertainties The direct and indirect life cycles of R. nana were tested were resolved by testing the infectivity of a number of in this study using eggs and cysticercoids sent by Dr Ito human isolates of R. nana in specific pathogen free (SPF) (Gifu University, Japan). Infected Tribolium confusum sent mice, rats and hamsters under varying conditions. by Dr Ito were killed and dissected using a WILD M7A

Table 2. Cortisone acetate treatment regime in mice, rats and hamsters prior to inoculation with human isolates of Rodentolepis (=Hymenolepis) nana.

SPF strain Species Number Sex Age Cortisone treatment Adult worms obtained

Swiss Qa Mouse 3a/3* Male 4±5 wks 125 mg kg21; 5 dosesa 0 AJa Mouse 3a/3* Male 4±5 wks 125 mg kg21; 5 dosesa 0 CBA/CAHa Mouse 3a/3* Male 4±5 wks 125 mg kg21; 5 dosesa 0 AJb Mouse 3b/3* Male 4±5 wks 125 mg kg21; 5 dosesb 0 CBA/CAHb Mouse 3b/3* Male 4±5 wks 125 mg kg21; 5 dosesb 0 Fischer 344c Rat 3c/3* Male 4±5 wks 100 mg total in 2 dosesc 0 Wistarc Rat 3c/3* Male 4±5 wks 100 mg total in 2 dosesc 0 DSNd Hamsters 15d Male 8 wks 25 mg kg21 in 2 dosesd 0 a Cortisone injections days 2, 4, 6, 8, 10, 12 (dose-weight dependent). b Cortisone injections days -1, 1, 3, 5, 7, 9 (dose-weight dependent). c Cortisone injections days 4 and 6 (dose-weight independent). d Cortisone given to all 15 hamsters days -1 and 4. * Control group received no cortisone. Infectivity of human R. nana to rodents 39 stereo microscope (WILD Heerbrugg, Switzerland). use of a congenitally hypothymic mouse model (Ito, Cysticercoids were collected into phosphate buffered 1985). saline (PBS) and orally inoculated, via stomach tube, into 6. Feeding NaCl-extracted eggs to Tribolium confusum Swiss Quackenbush (Swiss Q) and BALB/c male, 4- to 5- (flour beetles) that were pre-starved for 6 days (Naka- week-old mice. Approximately 11 days later, adult mura & Okamoto, 1993) on either filter paper (Yan & worms were dissected from the and Norman, 1995) or a latex rubber sheet (Nakamura & washed repeatedly in PBS. Eggs were teased from the Okamoto, 1993). gravid proglottids from worms obtained in this manner 7. Feeding T. confusum (pre-starved for 6 days) directly on and routinely passaged through mice and beetles. In infected faeces (Pappas et al., 1995; Yan & Norman, 1995). addition, R. nana eggs sent by Dr Ito were stored in PBS at 48C and were periodically inoculated into mice (19, 23, 26, 62 and 70 days post-collection). This was primarily to Cortisone acetate treatment provide a positive control of the decline of viability of eggs stored at non-ambient temperatures over a several Five different strains of SPF mice were used for the week period. When mice were inoculated with eggs, experimental procedures including Swiss Q (Thompson, rather than cysticercoids, euthanasia and dissection were 1972; Seidel & Voge, 1975) BALB/c (Asano et al., 1986; delayed until day 14 due to the longer pre-patency of Watanabe et al., 1994; Matsuo & Okamoto, 1995); A/J and infection. CBA/CAH (Ito et al., 1988); two strains of SPF rats: Fischer 344 (Ito, 1983) and Wistar, an equivalent strain to the Rowett rats used by (Ito & Kamiyama, 1987) and SPF Infection of mice, rats and hamsters using human samples hamsters (Ronald & Wagner, 1975). The SPF mice and rats used in this study (tables 1 and 2) were purchased from the Resources Centre Mice (ARC), Western Australia. The infection trials were Six Swiss Q, A/J and CBA/CAH mice were divided conducted at Murdoch University, Western Australia. into two equal groups. All mice were weighed prior to The hamster infection trial (table 2) was conducted by the trial to adjust their dose of cortisone accordingly. A J.M. Behnke with a human isolate of R. nana from our sub-cutaneous injection of cortisone acetate (Sigma, USA) collection. At Murdoch University, R. nana eggs were at a dose of 125 mg kg21 (Malinverni et al., 1995) was suspended in 0.1 ml phosphate buffered saline, drawn given to half the group on days 2, 4, 6, 8, 10 and 12 into a 1 ml hypodermic syringe fitted with a blunted 23 (treatmenta, table 2). The remaining half of each group gauge needle and 0.5 mm polyethylene tubing then were used as controls and did not receive any cortisone orally inoculated into rodent hosts. treatment prior to inoculation with R. nana eggs. All six mice in each group were inoculated with 2000 R. nana eggs in 0.2 ml PBS on day 0, regardless of their status Infection methods tested using human isolates (controls and test). A slightly modified regime (treat- b A variety of egg collection and inoculation methods ment , table 2) was also tried using A/J and CBA/CAH were tested using human isolates of R. nana in both mice mice whereby mice received their first cortisone dose and beetles: 24 h prior to inoculation (day -1), followed by five subsequent doses on days 1, 3, 5, 7 and 9. All mice, 1. Oral inoculation of mice, via stomach tube, using eggs regardless of their status, were killed and dissected 14 extracted by NaCl flotation. The size of each inoculum days post-inoculation. ranged from 50 to 14 400 eggs (Ferretti et al., 1980; Ito, 1982, 1984; Palmas et al., 1984). Rats 2. Oral inoculation of mice, via stomach tube, using eggs Six each Wistar and Fischer 344 rats were divided into extracted by Ficoll/sodium diatrizoate gradient centrifu- two equal groups. Half of each group received a sub- gation (Lumb et al., 1988) and sucrose gradient methods cutaneous injection of 50 mg cortisone acetate on days 4 outlined by Berntzen & Voge (1962), Lethbridge (1971) and 6 (Ito & Kamiyama, 1987). All rats (controls and test) and Hurd & Burns (1994). were inoculated with 2000 R. nana eggs in 0.2 ml PBS on 3. Oral inoculation of mice, via stomach tube, using eggs day 0. All rats were killed and dissected 14 days post- treated by mechanical and chemical `cracking' with a inoculation. The same human sample was used to infect glass bead and trypsin digestion method outlined by Ito all mice and rats used in the cortisone treatment trial, (1975) except that the trypsin (ICN Biomedicals, Ohio, thus eliminating random `between-sample' variability USA) was dissolved in Tyrodes solution (Sigma, St Louis, from the experimental procedure. Missouri, USA) instead of Hanks' balanced salt solution and the glass beads (Sigma, USA) used were smaller Hamsters (425±600 mm). Fifteen hamsters were divided equally into five 4. Spiking of mouse food with eggs for ad libitum feeding groups. Although it was not possible to use the sample (Ferretti et al., 1980) except that bread pieces were used inoculated into the mice and rats, a single human sample instead of ground food pellets provided by the small was used to inoculate all 15 hamsters. All hamsters animal facility, Murdoch University. received a subcutaneous injection of cortisone acetate 5. The pharmacological immunosuppression (Lucas et al., (Sigma, USA) at a dose of 25 mg kg21 24 h prior to 1980) of mice, rats and hamsters using subcutaneous stomach tubing and again on day 4 post inoculation. injections of cortisone acetate (Sigma, USA) as well as the Each group was inoculated with varying numbers of 40 M.G. Macnish et al. eggs. Group 1 received approximately 1860 eggs each; infected with cysticercoids upon dissection 3 weeks post- group 2 received 2220; group 3 received 3260 eggs; group feeding. When mice were inoculated with cysticercoids 4 received 14 400 eggs and group 5 received 400 eggs. from beetles sent from Japan, adult worms were One hamster per group was killed on day 4 to examine consistently obtained whether 3, 4, 6, 7, 10 or 13 the intestine for cysticercoids, another on day 7 to look cysticercoids were used (data not shown). for immature worms and the remaining hamster on day 14 to look for mature adult worms. Infection trials of human isolates in mice and rats Viability tests Of 51 human faecal samples tested in mice and 24 samples cross-tested in beetles, cysticercoid development Trypsin digestion occurred in T. confusum with one sample only (table 1). Egg shells were removed from R. nana both mechani- This faecal sample was stored at 48C for 23 days prior to cally and chemically using a modified trypsin digestion egg extraction using NaCl flotation then fed to beetles. method (see point 3, infection methods tested using Five beetles harboured 73 cysticercoids in total (table 1). human isolates). Two Swiss Q mice were inoculated with 13 and 25 cysticercoids respectively within 2 h of dissection from Nucleic acid dyes the beetles. Two more Swiss Q mice were inoculated Nucleic acid dye staining techniques used successfully three days later with 13 and 22 cysticercoids respectively for viability testing of Giardia and Cryptosporidium spp. that had been stored at 48C since collection. No adult by other researchers (Schupp & Erlandsen, 1987; Belo- worms developed from these cysticercoids in all four sevic et al., 1997), were evaluated for their indicator of mice (table 1). All remaining human samples failed to viability of R. nana eggs extracted from human faeces. yield either cysticercoids in beetles or adult worms in Stock and working solutions of fluorescein diacetate mice, including those inoculated into the hypothymic (FDA) and propidium iodide (PI) (Sigma, USA) were BALB/c mice regardless of the methods used for egg prepared according to the method outlined by Schupp & collection and inoculation of the hosts (table 1). Erlandsen (1987) whilst SYTO-9 and hexidium iodide (HI), from the BacLighte Bacterial gram strain kit (Molecular Probes, Eugene, Oregon, USA), were pre- pared according to manufacturers instructions. A pellet Infection trial of mice, rats and hamsters treated with cortisone of R. nana eggs was obtained by centrifugation at full acetate speed for 30 s and excess supernatant removed. Giardia All mice and rats inoculated with the same human cultures and Cryptosporidium oocysts, maintained routi- isolate were not infected with adult worms when nely in our laboratory, were used as controls (positive dissected 14 days post-inoculation regardless of whether and negative) and R. nana eggs, heated to 708C for treated with cortisone acetate or not (table 2). Hamsters, 30 min, were also used as a negative control. Nucleic acid from each group, killed and dissected on day 4 post- staining using FDA and PI was carried out on ice for infection failed to yield cysticercoids from intestinal 10 min (Schupp & Erlandsen, 1987) as well as at 378C for scrapings. Furthermore, hamsters dissected on days 7 30 min. Nucleic acid staining using SYTO-9 and HI was and 14 were not infected with immature or mature carried out for 10, 30 and 60 min at 378C based on the worms respectively (table 2). method outlined by Belosevic et al. (1997). A sample of R. nana not exposed to nucleic acid dye was also used to check for intrinsic autofluorescence, a phenomenon Viability tests using trypsin digestion and nucleic acid dye found to occur in some cells (Steinkamp et al., 2000). staining Slides of all test and controls were viewed using a confocal laser microscope (BioRad, California, USA) Microscopical examination of aliquots of eggs exam- using 488 nm laser at 1% power. A 522Df35 filter was ined after trypsin digestion revealed many R. nana eggs used for FDA and SYTO-9 detection and a 680DF32 filter with hatched oncospheres (results not shown). When was used for PI and HI detection. inoculated into mice or fed to beetles, however, no development to cysticercoid stage or adult worm occurred. The use of nucleic acid dyes, FDA, PI, SYTO- Results 9 and hexidium iodide failed to provide an indication of viability of R. nana eggs for two reasons. Firstly, it was Infection trials using a laboratory reference isolate in mice and apparent that R. nana eggs are mildly autofluorescent in beetles the absence of the nucleic acid dyes (results not shown). Oral inoculation of eggs, stored at 48C, of the Japanese Secondly, it was found that heat-killed R. nana eggs reference strain yielded adult worms from both Swiss Q exhibited fluorescence with all four nucleic acid dyes and BALB/c mice when dissected 14 days post inocula- (results not shown) rendering the system unworkable for tion on all occasions (after 19, 23, 26, 62 and 70 days viability discrimination. The protozoan controls used in storage) providing a positive control of viability over each system (FDA/PI and SYTO-9/HI) worked accord- time (data not shown). Eggs from Dr Ito's isolate were ing to the published methods outlined, indicating the not tested for viability beyond day 70. Tribolium confusum failure to discriminate between heat-killed and non- beetles, starved for 6 days, then fed on terminal gravid treated R. nana eggs did not relate to the dyes themselves, proglottids obtained from these worms were consistently or a technical failure of the method. Infectivity of human R. nana to rodents 41

Discussion stage in beetles in this study was extracted using saturated NaCl flotation. The failure to infect mice, rats and hamsters with the Berntzen & Voge (1962) first suggested that the human isolates of R. nana collected in Western Australia preparation of `shell-free' R. nana eggs increases their is consistent with the hypothesis that `strains' or infectivity and this has been used routinely for obtaining `variants' of R. nana that are host adapted exist in eggs for inoculation into beetle and rodent hosts (cf. Ito, Australian communities. In an attempt to address this 1977, 1982, 1984). In our laboratory the egg shells were hypothesis, a number of variables were examined in the removed both mechanically and chemically using the current study including the use of three different rodent methods outlined by these researchers with some species (mice, rats and hamsters) together with an insect modification (see point 3 ± Infection methods tested model, supported by large sample sizes in each case. using human isolates). Despite observing many R. nana Numerous approaches were tested for their suitability in eggs with hatched oncospheres, these did not develop to the infection of the animal models, including the use of the cysticercoid stage or adult worm when fed to beetles several strains, and genetically and pharmacologically or inoculated into mice. modified . The numbers of cysticercoids inoculated into mice in Overall, considerable care was taken in handling the our study were consistent with other researchers who eggs to avoid all obvious treatments that could have have reported successful infection to adult worm stages accounted for poor infectivity. Faecal samples were kept with 1±20 cysticercoids (Lucas et al., 1980; Ito et al., 1988; cool and analysed as soon after collection as was Kumazawa, 1992). Furthermore, in the present study, the practicable. The potential for variation in infectivity consistent development of cysticercoids, obtained from rates due to the age of the sample was carefully the Japanese control isolate, into adult worms when addressed by using samples that were wide ranging in inoculated into laboratory rodents indicated that the age. The human samples collected in our survey were storage of cysticercoids in PBS prior to inoculation did always sealed in collection pots, usually immediately, but not adversely affect the viability of the cysticercoids. This always within a few hours of collection and stored on ice is consistent with the methods outlined by Nakamura & or refrigerated at 48C, thus decreasing the risk of long Okamoto (1993) who store their cysticercoids in physio- periods of exposure to air, humidity and high tempera- logical saline. tures. Quality control included concurrent experiments Based on previous research (Lucas et al., 1980; Ito et al., with a Japanese isolate of up to 70 days of age (stored at 1988; Asano et al., 1992; Maki & Ito, 1998) several 48C) which yielded adult worms when inoculated into different susceptible rodent hosts were used in the mice on all occasions, suggesting that eggs remain viable inoculation trials in this study. However, the use of for long periods if storage conditions are appropriate. In these highly susceptible mice, even when further this study, oral inoculation of eggs into mice and beetles, immunosuppressed with cortisone, and the use of including the direct feeding of faeces to beetles, was hypothymic rodent hosts did not result in the develop- carried out using human faecal samples that ranged in ment of eggs to either cysticercoid or adult worm stages age from 1 h (beetles only) through to 72 days old (mice in vivo. and beetles). Cysticercoids did not develop in the beetles The lack of development of 23 of the 24 human in all but one sample, nor did adult worms develop in samples of R. nana eggs tested in T. confusum beetles to any rodent host, regardless of their immune and genetic cysticercoid stage in this study is unlikely to be status. The development of R. nana eggs from one human attributable to factors such as variability in the genetics, sample to cysticercoid stage in T. confusum is a positive sex and nutritional status of the beetle host. Given that indication of viability. Reports in the literature regarding Yan & Norman (1995) found that two beetle species, egg viability are contradictory. Maki & Yanagisawa T. confusum and T. castaneum, varied in their suscept- (1987) suggest that the infectivity of eggs decreases ibility to H. diminuta as well as showing significant after 17 h exposure to air. In contrast, Pawlowski (1984) among-strain and between-sex variation, only one report a survival of eggs in the environment up to 10 species, T. confusum, was used in our study at all times. days. Kennedy (1983) found that eggs of H. diminuta can In addition, there is evidence that male beetles harbour survive and remain infective in rat faeces for up to 60 significantly greater numbers of cysticercoids than days when conditions are optimal. female beetles when infected with H. diminuta eggs The extraction techniques employed in this study are (Pappas et al., 1995). Whilst the sex of the beetles unlikely to have damaged the R. nana eggs. Although the employed in this study was not determined, the strain use of NaCl concentration has been criticized by some is routinely used in our laboratory for the maintenance of researchers who believe the procedure may reduce the H. diminuta suggesting that the sex ratio of males to infectivity of R. nana eggs (Yan & Norman, 1995; Ferretti females is sufficient for routine passage of this parasite. et al., 1980), Thompson (1972) repeatedly succeeded in Furthermore, this strain of beetle is routinely starved for infecting albino mice with R. nana eggs obtained by NaCl 4±5 days prior to inoculation with H. diminuta eggs in flotation and other researchers routinely use NaCl our laboratory. In experiments conducted with R. nana, extraction procedures for obtaining R. nana eggs for however, beetles were routinely starved for 6 days, as testing in mice (Nakamura & Okamoto, 1993). In proposed by Nakamura & Okamoto (1993), suggesting addition, Hurd & Burns (1994) used a combination of the failure to demonstrate infection with R. nana in saturated NaCl and sucrose to extract H. diminuta eggs beetles in this study was not due to insufficient from faeces and successfully infected laboratory rats. starvation times. The failure of human isolates to develop Moreover, the only sample which developed to cysticercoid to cysticercoid stage in beetles may, therefore, be 42 M.G. Macnish et al. explained by a progressive loss of adaptation of the Belosevic, M., Guy, R.A., Taghi-Kilani, R., Neumann, Australian human isolates to these intermediate hosts. In N.F., Gyurek, L.L., Liyanage, L.R.J., Millards, P.J. & light of this suggestion, the development of one of the 24 Finch, G.R. (1997) Nucleic acid stains as indicators of human samples to cysticercoid stage in beetles is difficult Cryptosporidium parvum oocyst viability. International to explain. Journal for Parasitology 27, 787±798. It is not entirely clear whether rodent species play a Berntzen, A.K. & Voge, M. (1962) In vitro hatching of role in the transmission of R. nana in Australia or oncospheres of Hymenolepis nana and H. citelli. Journal whether they harbour their own variant species/strain. of Parasitology 48, 110±119. In view of the reliability of the experimental protocols Crompton, D.W.T. (1999) How much human helminthia- being controlled by the use, in parallel, of eggs and sis is there in the world? Journal of Parasitology 85, 397± cysticercoids from a mouse strain of R. nana combined 403. with the use of a large sample size in this study, the Czaplinski, B. & Vaucher, C. (1994) Family Hymeno- failure to infect mice, rats and hamsters with 51 human lepididae Ariola, 1899. pp. 595±663 in Khalil, L.F., isolates of R. nana provides supportive evidence towards Jones, A. & Bray, R.A. (Eds) Keys to the cestode parasites the hypothesis that a human variant of R. nana exists in of vertebrates. Wallingford, CAB International. Australia. Ferretti, G., Gabriele, F. & Palmas, C. (1980) Methodol- A poor understanding of the dynamics of transmission ogy in experimental infections of mice with Hymeno- of a parasite undermines both short and long term lepis nana. Bollettino di Zoologia 47, 165±184. control strategies which can only be effective when Holland, C.V., O'Shea, E., Asaolu, S.O., Turley, O. & accurate epidemiological information is obtained (Hol- Crompton, D.W.T. (1996) A cost-effectiveness analysis land et al., 1996). Identifying the risk factors within of anthelminthic intervention for community control of communities, from detailed epidemiological data, is soil-transmitted helminth infection: levamisole and likely to be the most cost efficient for implementing . Journal of Parasitology 82, 527±530. control strategies (WHO, 1996). 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